The T-cell receptor (TCR) and its coreceptor CD8 are used by cytotoxic T lymphocytes (CTL) to identify virus-infected or malignant target cells. Such target cells express class I MHC molecules with bound peptide ligands that are specifically recognized by the TCR/CD8 complex. The work proposed here will investigate several aspects of the molecular interaction of TCR/CD8 with class I/peptide. It has been recently determined that a single peptide is immunodominant in Ld- allorecognition. This observation will be extended to determine if this immunodominance is reflected in the quantitative expression of this peptide, the affinity of the peptide for Ld, or the affinity of the TCR for the Ld/peptide complex. As an extension of earlier studies, this laboratory has developed a method to generate peptide-specific alloreactive CTL to H-2Ld. Using this methodology, peptide-specific alloreactive CTL will be generated to known endogenous Ld ligands to determine whether they function in allogeneic responses and whether their expression is ubiquitous on different cell types. We will also test whether allogeneic CTL, specific for endogenous Ld ligands, are more peptide cross-reactive than syngeneic CTL specific for viral peptide ligands. To better understand how specific TCR structural motifs interact with class I or its bound ligand, an extensive panel of site-specific mutants of class I will be subjected to structure-function analyses. These studies will also include several CTL clones restricted by the same class I but specific for different peptides as well as CTL clones restricted by similar class I molecules and specific for the same peptide. As an extension of earlier studies mapping the CD8 recognition site, a class I alpha-3 mutant not recognized by CD8, will be characterized in vivo and in vitro to better define the role of CD8 in the induction of CTL responses and in class I recognition by CTL. In summary, the proposed experiments will exploit unique resources and approaches to define various parameters governing functional interactions of the TCR/CD8 molecules on CTL with class I/peptide molecules on target cells.